Parallel G-Quadruplex DNA Structures from Nuclear and Mitochondrial Genomes Trigger Emission Enhancement in a Nonfluorescent Nano-aggregated Fluorine–Boron-Based Dye

Molecular self-assembly is a powerful tool for the development of functional nanostructures with adaptive optical properties. However, in aqueous solution, the hydrophobic effects in the monomeric units often afford supramolecular architectures with typical side-by-side π-stacking arrangement with compromised emissive properties. Here, we report on the role of parallel DNA guanine quadruplexes (G4s) as supramolecular disaggregating–capture systems capable of coordinating a zwitterionic fluorine–boron-based dye and promoting activation of its fluorescence signal. The dye’s high binding affinity for parallel G4s compared to nonparallel topologies leads to a selective disassembly of the dye’s supramolecular state upon contact with parallel G4s. This results in a strong and selective disaggregation-induced emission that signals the presence of parallel G4s observable by the naked eye and inside cells. The molecular recognition strategy reported here will be useful for a multitude of affinity-based applications with potential in sensing and imaging systems.


S3
Procedure for the preparation of substituted benzimidazole-iminocoumarin compound (3a-b): To the mixture of benzimidazole-iminocoumarin 2 (400 mg, 1.12 mmol) and corresponding aniline (300 mg, 2.5 mmol) in 20 mL of ethanol, catalytic amount (18 mg, 10 mol%) of p-TsOH was added and the resulting mixture was refluxed overnight. Completion of reaction was monitored through LC-MS. On completion, solvent was cooled and then removed under reduced pressure. The crude so obtained was purified by column chromatography over silica (SiO2), using 0-1.5% methanol in dichloromethane as eluent. was obtained from the reaction of 2 (400 mg) with 4-morpholinoaniline (440 mg) by following the general procedure as a red solid in 69% (401 mg) yield. 1

S4
General procedure for preparation of benzimidazole-iminocoumarin based bodipy complexes (4a-b): To the mixture of benzimidazole-iminocoumarin (300 mg, 0.69 mmol) in dichloroethane, was added diisopropylamine (0.3 mL, 1.7 mmol) and BF3.OEt2 (0.23 mL, 1.7 mmol) and the resulting mixture was stirred at 90 °C for 1-2 hours. Completion of the reaction was monitored through LC-MS. On completion, the reaction mixture was allowed to attain room temperature.
The cooled mixture was diluted with dichloromethane and washed with a saturated solution of NaHCO3. The organic layers were combined, dried over MgSO4 and the solvent was removed under reduced pressure. The crude mixture so obtained was purified through column chromatography in silica (SiO2) with 0-2% methanol in dichloromethane as eluent.

Experimental procedures General
Solvents, reagents, chemicals and oligonucleotides were purchased from commercial suppliers (Sigma-Aldrich and Eurofins Genomics) and used without further modifications, unless otherwise stated. Oligonucleotides were diluted with ultrapure water and stored at -20 °C. The exact oligonucleotide concentration was determined by UV/Vis spectroscopy using the molar extinction coefficients (ε260) provided in Table S1

Optical spectroscopy
CD measurements were recorded on a Jasco J-1700 CD spectrometer at 25 °C using a cell length of 1 cm, and over a wavelength range of 220-320 nm. CD spectra were baseline-corrected from the buffer. UV/Vis absorption spectra were recorded on T90+ UV/Vis spectrometer (PG instruments Ltd). The spectral band width was 1 nm. Steady-state emission spectra and kinetic curves were recorded on Jasco FP-6500 spectrofluorometer equipped with the JascoPeltier-type temperature controller (ETC2736).

Atomic force microscopy (AFM)
AFM imaging was carried out by using a BioScope Catalyst atomic force microscope (Bruker) in peak force tapping mode in air. Resolution was set at 512 × 512 pixels, scan size was 1 μm, and scan rate was 1.0 Hz. Peak force set point and gain were controlled automatically. Bruker ScanAsyst-Air cantilevers were used in all measurements. Samples were deposited on the S11 surface of freshly cleaved mica (Ted Pella) for 15 min, washed three times with 100 μL deionized water, and dried at room temperature.

Dynamic Light Scattering (DLS)
DLS was performed by using a MAVERN particle size analyzer Nano S instrument. Samples were prepared at 1 μM concentration by diluting them from the DMSO stock solutions into water (DMSO 0.1 % and 0.2 % for 4b and 4a, respectively). Before analysis the samples were left to equilibrate for 2 hours. Particle size is provided as the average of three independent measurements.

Photometric and fluorimetric titrations
The freshly prepared 4b solution (c4b = 3 μM, cTRIS = 50 mM, pH: 7.4, cKCl = 100 mM) was titrated with the oligonucleotides and let to equilibrate for several minutes before recording the UV/Vis or emission spectra. The concentration of the experiments was optimized to have an optical density < 0.15 to avoid reabsorption in the fluorescence emission. The excitation wavelength was set at the isosbestic point centered at 513 nm.

Light-up response of 4b complexed with mitochondrial DNA G4 sequences
The freshly prepared 4b solution (c4b = 1 μM, cTRIS = 50 mM, pH: 7.4, cKCl = 100 mM) was titrated with the mtDNA G4s (see Table S3) (cG4s = 2 μM) and let to equilibrate for 30 minutes before recording the emission spectra. The excitation wavelength was set at the isosbestic point centered at 513 nm. Fold change in fluorescence intensity is defined as the ratio between 4bmt DNA G4 system (F) over 4b emission intensity (F0).

Fluorescence displacement assay
Fluorescence displacement assay was performed by titrating the 4b-c-KIT 2 binary mixture with increasing concentration of the well-known G4-binders Phen-DC3 or BRACO-19 until no change on the 4b´s fluorescence intensity was observed.

Data processing
Binding constants were obtained with Bindfit 1, 2 by using multiple global fitting methods  Table S2.

Solvent-dependent studies
Solvent-dependent studies were performed by dissolving 4a or 4b in different organic solvents having different polarities. Then, the UV/Vis and the emission spectra were recorded by exciting the samples at their absorption maxima.

Aggregation studies
Disaggregation studies were carried out by titrating sodium dodecyl sulfate (SDS) into 4a or have a significant effect on the G4 structure. All spectra were recorded in 3 mm NMR tubes at S13 298 K on a Bruker 850 MHz Avance III HD spectrometer equipped with a 5 mm TCI cryoprobe.
Excitation sculpting was used in the 1 H NMR experiments, and 256 scans were recorded.

Cytotoxicity assay
HeLa cells were culture at 37 o C in 5% CO2 in DMEM medium supplemented with penicillinstreptomycin (1×), and 10% fetal bovine serum. 5 × 10 3 cells/well were seeded in complete medium on 96 well-plates 24 h before the treatment with 4b. 4b was dissolved in complete  was incubated with BRACO-19 (5, 10 or 20 μM) for 30 min at RT. S14 Images were acquired using a Leica SP8 FALCON confocal microscope equipped with an incubation chamber operating at 37 °C in 5% CO2. Maximum intensity projection of Z-stack images was used for data presentation. All data were processed by using ImageJ software.   Table S1.

UV/Vis titration studies of 4a in the presence of parallel and hybrid G4s
Figure S11. UV/Vis absorption spectra of 4a in the absence (blue line) and presence (gray and red lines) of increasing concentrations of parallel and hybrid G4s (c4a = 3 μM, cparallel/hybrid G4s = 0 to 17 μM, cKCl = 100 mM, cTRIS = 10 mM, pH: 7.4). The sequences of the tested G4 oligonucleotides are shown in Table S1.  Table  S2.